CN103792359A - Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit - Google Patents

Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit Download PDF

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CN103792359A
CN103792359A CN201210435492.8A CN201210435492A CN103792359A CN 103792359 A CN103792359 A CN 103792359A CN 201210435492 A CN201210435492 A CN 201210435492A CN 103792359 A CN103792359 A CN 103792359A
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杜道林
王永美
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Jiangsu Wise Science and Technology Development Co Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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Abstract

The invention discloses an enzyme-linked immunosorbent assay kit for detecting aflatoxin G1 in peanuts, cereals and milk products. In the kit, based on a competitive ELISA (enzyme-linked immunosorbent assay) method, ELISA plate micropores are coated with the aflatoxin G1 antigen. The detection comprises the following steps: adding a standard product or sample solution, an aflatoxin G1 antibody and an enzyme marker; competitively combining the coating antigen and the aflatoxin G1 added into the sample with the aflatoxin G1 antibody; washing off the uncombined antigen and antibody; further combining the combined antigen-antibody compound with the enzyme marker; washing off the uncombined antigen and antibody, and developing color through a TMB substrate; adding a reaction stop solution, and detecting with a 450/630nm dual-wavelength microplate reader, wherein the aflatoxin G1 concentration in the sample is inversely proportional to the absorption light intensity; and comparing with the standard curve and multiplying with corresponding dilution ratio to obtain the content of aflatoxin G1 in the sample.

Description

The preparation of aflatoxin G 1 enzyme-linked immunologic detecting kit and detection method
Technical field
Detect enzyme-linked immunoassay kit and the detection method of aflatoxin G 1, belong to biology and food safety detection technical field, can be used for the detection to aflatoxin G 1 (AFG1) content in peanut, cereal, dairy produce.
Background technology
Aflatoxin is mainly the secondary metabolite being produced by aspergillus flavus and aspergillus parasiticus secretion, is a kind of natural toxic compounds that can cause the various infringements of people and animals.Aflatoxin (AFT) has found that more than 20 plant at present, and wherein aflatoxin B1 is toxicity and the strongest material of carcinogenicity.The toxicity of aflatoxin G 1 is only second to aflatoxin B1, is a kind of strong carcinogen.There are some researches show, it has the stronger potentiality of bringing out tumor of kidney than AFB1.One of main contaminant toxin in Ta Shi China the southern segment of Taihang Mountain cancer of the esophagus, Area of High Lung Cancer Incidence (as Ci County, Hebei province) resident's diet, its recall rate and content are all quite high.
Figure 748410DEST_PATH_IMAGE001
aflatoxin G 1 structural formula
It is the problem that China's food export frequently runs into that aflatoxin exceeds standard, and the annual food export of China suffers circular to detain generation repeatedly because aflatoxin exceeds standard.Control the content of aflatoxin in varieties of food items and be more and more subject to national attention.To aflatoxin, the residue limits in food all has regulation in various countries.European Union in council's regulations (EC) No 629/2008, council's regulations (EC) No 1881/2006 to aflatoxins the limitation in the food such as peanut, cereal, nut make clearly regulation.The U.S. comprises that at animal feed, food, milk, peanut and goods thereof, nut the limitation in Bertholletia excelsa, pistachio nut makes stipulations to aflatoxin in FDA " observe Statutes Compliance Policy Guide ".Japan in Positive List System in regulation food the limitation of aflatoxin be 10 ppb.
The method of measuring at present aflatoxin mainly contains thin-layered chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, mass spectroscopy, radioimmunoassay.These detection methods have been brought into play good effect at different times, but owing to more or less existing complicated operation, polluting the defects such as large, high to instrument and equipment requirement, false positive rate is high, detection time is long, have been difficult to adapt to society requirement.For appeal problem, the invention provides aflatoxin G 1 enzyme-linked immunoassay kit, this kit have pollute little, highly sensitive, detection time is short, high specificity be applicable to the testing requirement of great amount of samples, be with a wide range of applications.
Summary of the invention
The present invention is that the Enzyme-linked Immunosorbent Assay of aflatoxin G 1 detects dedicated kit, for the detection to peanut, cereal, dairy produce aflatoxin G 1 (AFG1) content.Its detection is quick, sensitive, accurate, can be quantitative, easy and simple to handle, and less demanding to sample purity, high specificity, be specially adapted to the detection of batch samples, for this reason, enzyme-linked immunoassay kit and detection method that the present invention also provides special agent to detect aflatoxin G 1 comprise:
(1) the coated plate in 96 of polystyrene material or 48 holes;
(2) aflatoxin G 1 standard items, concentration is 0 ng/mL, 0.025 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.5 ng/mL dilute and obtain from AFG1 sterling, and dilution is methyl alcohol: water volume ratio is 4:6;
(3) AFG1-BSA: MCPBA (85%, 9.86 mg) is dissolved in 1 mLCH 2cl 2in, wash four times each 2 mL with Pi/NaCl damping fluid (0.1 mol/L, pH7.0).2 mg AFG1 dry powder are dissolved in to 1 mL CH 2cl 2in, add 2 mLPi/NaCl damping fluids (0.1 mol/L, pH7.2), with the CH of MCPBA 2cl 2solution mixes, and 25 ℃, magnetic agitation 80 min.Discard upper strata water, the Pi/NaCl that contains BSA13.6 mg (pH7.2) solution is added, vigorous stirring 4 h under room temperature.Centrifugal 20 min of 4000 r/min, get upper strata water, use CH 2cl 2wash three times, in Pi/NaCl (pH7.4) dialysis 3 d, sooner or later change liquid once.Freeze drying, obtains AFG1-BSA compound;
(4) monoclonal antibody of aspergillus flavus resisting toxin G1: take AFG1-BSA as immunogene, adopt the immunization method immunity small white mouse of back multi-point injection, in triplicate.The 14th d from injecting immune is for the first time former starts, take a blood sample once every 14 d, separation of serum, measure absorbance with indirect non-competing ELISA, take absorbance as ordinate, sample interval week number is horizontal ordinate, obtains antibody and produces process curve, and result shows that three mouse all can produce antibody preferably.
Figure 562782DEST_PATH_IMAGE002
              
(5) the goat anti-rabbit antibody dried frozen aquatic products (HRP-goat anti-rabbit antibody) of horseradish peroxidase-labeled;
(6) cleansing solution: ten hydrogen phosphate dihydrate sodium 68.8 g add sodium dihydrogen phosphate 6.9 g, sodium chloride 45 g, Tween-20 0.5 mL, adds distilled water to 1 L, regulates PH to 7.4;
(7) nitrite ion A and nitrite ion B:A liquid are 0.2 M Na2HPO 425.7 the H of mL+0.1 M citric acid 24. 3 mL and 30% 2o 250 mL; Being formulated as of B liquid takes 5 mg tetramethyl biphenyl diamines (TMB) and adds in 2.5 mL absolute ethyl alcohols, can be heated to 37 ℃～40 ℃ until TMB dissolve completely;
(8) stop buffer is the sulfuric acid solution of 2 M.
It is characterized in that: get the coated plate of the coated micropore that has AFG1-BSA, the sample that adds AFG1 standard items and handle well is in micropore separately, add again HRP-goat anti-rabbit antibody, add again AFG1 antibody, oscillating reactions, cleansing solution washing, add nitrite ion A and nitrite ion B, after lucifuge reaction, add stop buffer, under microplate reader 450/630 nm dual wavelength, survey light absorption value, the AFG1 content in reference standard curve calculation sample.
Beneficial effect of the present invention: this kit is simple, easy to use, can detect great amount of samples, highly sensitive simultaneously, can reach 0.025 ng/mL.
Accompanying drawing explanation
Figure below is the canonical plotting of AFG1.
Embodiment
The enzyme-linked immuno sorbent assay kit of aflatoxin G 1, mainly comprises the coated plate in 96 or 48 holes, aflatoxin G 1 standard items, AFG1-BSA, the monoclonal antibody of aspergillus flavus resisting toxin G1, the goat anti-rabbit antibody dried frozen aquatic products of enzyme labeling, cleansing solution, nitrite ion A, nitrite ion B and stop buffer.
the pre-treatment of embodiment 1 experiment material and sample
Coated plate solid phase antigen preparation:
By 50 mmo1/L Na for AFG1-BSA 2c0 3-NaHC0 3pH9. 6 damping fluids are diluted to the coating buffer of 10 mg/L, and the each hole of microwell plate, 96 (or 48) hole adds 100 μ L, and lucifuge is hatched 2 h at 37 ℃, discards coating buffer, washs 2 times, add the above-mentioned Na of 150 μ L containing 3%BSA 2cO 3-NaHCO 3damping fluid sealing, lucifuge is hatched 1.5 h at 37 ℃, discards confining liquid, dries rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent:
(1) standard items aflatoxin G 1: (0 ng/mL, 0.025 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.5 ng/mL), from AFG1 sterling, diluting and obtain, dilution is methyl alcohol: water volume ratio is 4:6;
(2) the goat anti-rabbit antibody dried frozen aquatic products (HRP-goat anti-rabbit antibody) of horseradish peroxidase-labeled;
(3) cleansing solution: ten hydrogen phosphate dihydrate sodium 68.8 g add sodium dihydrogen phosphate 6.9 g, sodium chloride 45 g, Tween-20 0.5 mL, adds distilled water to 1 L, regulates PH to 7.4;
(4) nitrite ion A:0.2 M Na 2hPO 425.7 mL, 0.1 M citric acid 24. 3 mL and 30% H 2o 250 mL;
(5) nitrite ion B: take 5 mg tetramethyl biphenyl diamines (TMB) and add in 2.5 mL absolute ethyl alcohols, can be heated to 37 ℃～40 ℃ until TMB dissolve completely;
(6) stop buffer: the H of 2 mo1/L 2sO 4.
Reagent in each box enough carries out 96 test sample amounts, and the material in box is as follows:
(1) 96 orifice plate (8 × 12 hole, can be split as single hole) is coated with AFG1-BSA;
(2) 6 bottles of AFG1 titers, 1.0 mL/ bottles, concentration of standard solution is: 0 ng/mL, 0.025 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.5 ng/mL;
(3) AFG1 monoclonal antibody, by ELISA program, to the said monoclonal antibody test of tiring, the mouse antibodies that produces antibody tires in 450nm place absorbance to be 1 o'clock, to reach respectively 1:10000,1:10000 and 1:12000;
(4) HRP mono-goat anti-rabbit antibody dried frozen aquatic products, used time l0 mL distilled water dissolves;
(5) cleansing solution: 30 mL, the used time is with distilled water dilution in 1: 20;
(6) nitrite ion A:10 mL;
(7) nitrite ion B:10 mL;
(8) stop buffer: 10 mL.
In experiment, need the reagent of use
Methyl alcohol (analyzing pure), sherwood oil or normal hexane (analyzing pure), methenyl choloride or methylene chloride (analyzing pure), deionized water
Sample pre-treatments step is as follows:
(1) peanut: get the sample that 5 g peanut samples are pulverized, add 20 mL sherwood oils or normal hexane and 25 mL sample extracting solutions, fully vibration mixes 5-10 min, after centrifugal or stratification, remove supernatant liquid, get 0.2 mL lower floor liquid and join and in 1 mL sample diluting liquid, dilute and fully mix, get 50 μ L dilutions after sample to be measured;
(2) cereal: get 5 g grain samples and pulverize sample, add 25 mL sample extracting solutions, fully vibration mixes 5-10 min, centrifugal 5 min of 4000 r/min, or use quantitative test Filter paper filtering, get the supernatant of 0.2 mL after centrifugal or filter after filtrate join and in 1 mL sample diluting liquid, dilute and fully mix, get sample after 50 μ L dilute to be measured;
(3) milk powder: get 5 g milk powder in 50 mL centrifuge tubes, add 10 mL sample extracting solutions, thermal agitation 5 min, filter or centrifugal 5 min of 4000 r/min, get 1 mL supernatant, add the deionized water of 1.5 mL and sherwood oil or the normal hexane of 2.5 mL, vibration shakes up, centrifugal 5 min of 4000 r/min, transfer to lower floor's liquid in another clean centrifuge tube, for detection of.
embodiment 2 methods are measured
Points for attention before measuring
1, use before by all reagent with need go up to room temperature with microwell plate;
2, use and immediately all reagent is put back to 2 ~ 8 ℃ afterwards;
3, in use do not allow micropore dry;
4, the repeatability in elisa assay, depends on the consistance of washing plate to a great extent, and the correct plate operation of washing is the main points in ELISA mensuration program;
5, in all constant-temperature incubation processes, avoid light to irradiate, live microwell plate with cover plate membrane cover;
6, take out the microwell plate and the framework that need by quantity, no microwell plate is put in former Fresco Bag and together with the drying agent providing and resealed, be stored in 2-8 ℃.
Concrete determination step
1, required reagent and microwell plate are taken out from cold storage environment, at room temperature balance 30 min, must shake up before every kind of liquid uses.Notice that titer all needs to do 2 parallel experiments;
2, add standard items/sample: add standard items/sample 50 μ L in corresponding micropore, then add enzyme labeling thing 50 μ L/holes, and then add 50 μ L/ hole antibody working fluids, vibration mixes gently, react 30 min by cover plate membrane cover plate postposition room temperature lucifuge;
3, wash plate: carefully open cover plate film, liquid in hole is dried, add cleansing solution 250 μ L/ holes, soak 15 ~ 30 s at every turn, fully wash 4 ~ 5 times, pat dry with thieving paper;
4, colour developing: add substrate solution A liquid 50 μ L/ holes, then add substrate solution B liquid 50 μ L/holes, vibration mixes gently, with rearmounted 25 ℃ of lucifuge environment reaction 15 min of cover plate membrane cover plate;
5, measure: every hole adds 50 μ L stop buffers, set microplate reader at 450 nm places, measure OD value (suggestion 450/630 nm dual wavelength detects, and runs through data in 5 min).The mean value (B) of measured titer or sample absorbance is divided by the absorbance (B of first titer (0 titer) 0) value is multiplied by 100% again, obtains percentage absorbance.
Percentage absorbance (%)=B/B 0× 100%
Take the logarithm at 10 end of as of standard items concentration as X-axis, percentage light absorption value is Y-axis, drawing standard curve.By the percentage light absorption value substitution typical curve of sample, read the corresponding value of sample from typical curve, as 10 power, be multiplied by extension rate, be the amount of contained aspergillus flavus G1 in sample.
  

Claims (7)

1. for detection of the enzyme linked immunological kit of aflatoxin G 1, it is the coated plate (1) by 96 or 48 holes, aflatoxin G 1 standard items (2), AFG1-BSA(3), the monoclonal antibody (4) of aspergillus flavus resisting toxin G1, the goat anti-rabbit antibody (5) of enzyme labeling, cleansing solution (6), nitrite ion A (7), nitrite ion B(8) and stop buffer (9) form.
2. the antibody dried frozen aquatic products that the goat anti-rabbit antibody of described enzyme labeling is horseradish peroxidase-labeled, cleansing solution is the Tris-HCL damping fluid that contains tween, nitrite ion A is the citric acid-sodium hydrogen phosphate damping fluid that contains hydrogen peroxide, nitrite ion B is the ethanolic solution of tetramethyl biphenyl diamines, and stop buffer is the sulfuric acid solution of 2 M.
3. kit according to claim 1, the coated solid phase antigen of coated plate (1) wherein, with 50 mmol/LpH9.6 Na 2cO 3-NaHCO 3damping fluid AFG1-BSA is diluted to 10 mg/L as coating buffer, 96 holes or 48 hole microwell plates respectively add 100 μ L, lucifuge is hatched 2 h at 37 ℃, discards coating buffer, washs 2 times, adds the above-mentioned Na of 150 μ L containing 3 %BSA 2cO 3-NaHCO 3damping fluid sealing, lucifuge is hatched 1.5 h at 37 ℃, discards confining liquid, dries rearmounted-20 ℃ of freezing preservations of lath sealing.
4. according to the kit described in claims 1, aflatoxin G 1 standard items (2) wherein, from AFG1 sterling, dilute and obtain, dilution is methyl alcohol: water volume ratio is 4:6, totally 6 bottles, AFG1 concentration is respectively 0 ng/mL, 0.025 ng/mL, 0.1 ng/mL, 0.25 ng/mL, 0.5 ng/mL, 1.5 ng/mL.
5. detect the method for aflatoxin G 1 according to the kit described in claims 1, it is characterized in that getting the coated plate of the coated micropore that has AFG1-BSA, the sample that adds AFG1 standard items and handle well, in micropore separately, adds HRP-goat anti-rabbit antibody, then adds AFG1 antibody, oscillating reactions gently, cleansing solution washing, adds nitrite ion A and nitrite ion B, after lucifuge reaction, adds stop buffer, under microplate reader, survey light absorption value, the AFG1 content in reference standard curve calculation sample.
6. the method for survey aflatoxin G 1 according to claim 5, it is operating as: get the coated plate of micropore that is coated with AFG1-BSA, add the AFG1 standard items of 50 μ L and the sample handled well in micropore separately, add 50 μ L HRP-goat anti-rabbit antibodies, add again the anti-AFG1 antibody of 50 μ L, at 25 ℃, lucifuge is reacted 30 min, wash dropping liquid and wash plate 5 times, at 25 ℃, lucifuge is reacted 30 min, cleansing solution is washed plate 5 times, add nitrite ion A and nitrite ion B, at 25 ℃, lucifuge is reacted 15 min, add stop buffer, under 450/630 nm dual wavelength, measure light absorption value, AFG1 content in reference standard curve calculation sample.
7. the method for detection aflatoxin G 1 according to claim 5, sample treatment is wherein (1) cereal: get 5 g and pulverize sample, add 25 mL sample extracting solutions, fully vibration mixes 5-10 min, centrifugal 5 min of 4000 r/min, or use quantitative test Filter paper filtering, get the supernatant of 0.2 mL after centrifugal or filter after filtrate join and in 1 mL sample diluting liquid, dilute and fully mix, get sample after 50 μ L dilute to be measured; (2) peanut: get the sample that 5 g pulverize, add 20 mL sherwood oils or normal hexane and 25 mL sample extracting solutions, fully vibration mixes 5-10 min, after centrifugal or stratification, remove supernatant liquid, get 0.2 mL lower floor liquid and join and in 1 mL sample diluting liquid, dilute and fully mix, get 50 μ L dilutions after sample to be measured; (3) milk powder: get 5 g milk powder in 50 mL centrifuge tubes, add 10 mL sample extracting solutions, thermal agitation 5 min, filter or centrifugal 5 min of 4000 r/min, get 1 mL supernatant, add the deionized water of 1.5 mL and sherwood oil or the normal hexane of 2.5 mL, vibration shakes up, centrifugal 5 min of 4000 r/min, transfer to lower floor's liquid in another clean centrifuge tube, for detection of
CN201210435492.8A 2012-11-05 2012-11-05 Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit Pending CN103792359A (en)

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CN104569380A (en) * 2015-01-23 2015-04-29 天津伯克生物科技有限公司 Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit
CN105319351A (en) * 2014-07-24 2016-02-10 江苏维赛科技生物发展有限公司 Adamantanamine enzyme-linked immunosorbent assay detection special-purpose detection kit
CN106290821A (en) * 2016-08-09 2017-01-04 安徽青松食品有限公司 The authentication method of aflatoxin potential pollution in one peanut sugar
CN108663361A (en) * 2018-05-22 2018-10-16 福州大学 A kind of method of biomass in quick measurement liquid state fermentation liquid
CN111487403A (en) * 2019-01-28 2020-08-04 西北农林科技大学 Portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument and detection method
CN113607949A (en) * 2021-05-28 2021-11-05 中国农业科学院油料作物研究所 Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland
CN113721022A (en) * 2021-09-07 2021-11-30 中国农业科学院油料作物研究所 Method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof
CN113866421A (en) * 2021-09-07 2021-12-31 中国农业科学院油料作物研究所 Aflatoxin toxigenic fungi virulence indicator molecule rapid detection kit and application thereof

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105319351A (en) * 2014-07-24 2016-02-10 江苏维赛科技生物发展有限公司 Adamantanamine enzyme-linked immunosorbent assay detection special-purpose detection kit
CN104569380A (en) * 2015-01-23 2015-04-29 天津伯克生物科技有限公司 Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit
CN106290821A (en) * 2016-08-09 2017-01-04 安徽青松食品有限公司 The authentication method of aflatoxin potential pollution in one peanut sugar
CN108663361A (en) * 2018-05-22 2018-10-16 福州大学 A kind of method of biomass in quick measurement liquid state fermentation liquid
CN108663361B (en) * 2018-05-22 2020-12-25 福州大学 Method for rapidly determining biomass in liquid fermentation broth
CN111487403A (en) * 2019-01-28 2020-08-04 西北农林科技大学 Portable enzyme-linked immunosorbent assay (ELISA) sample liquid absorbance detection instrument and detection method
CN113607949A (en) * 2021-05-28 2021-11-05 中国农业科学院油料作物研究所 Method for rapidly identifying and comparing relative abundance of toxin-producing fungi of aflatoxin in farmland
CN113607949B (en) * 2021-05-28 2023-06-27 中国农业科学院油料作物研究所 Method for rapidly identifying and comparing relative abundance of toxigenic fungi of farmland aflatoxins
CN113721022A (en) * 2021-09-07 2021-11-30 中国农业科学院油料作物研究所 Method for rapidly identifying relative abundance of aflatoxin toxigenic bacteria in farmland and application thereof
CN113866421A (en) * 2021-09-07 2021-12-31 中国农业科学院油料作物研究所 Aflatoxin toxigenic fungi virulence indicator molecule rapid detection kit and application thereof
CN113721022B (en) * 2021-09-07 2023-06-27 中国农业科学院油料作物研究所 Quick identification method for relative abundance of aflatoxin-producing bacteria in farmland and application thereof
CN113866421B (en) * 2021-09-07 2023-06-27 中国农业科学院油料作物研究所 Rapid detection kit for virulence indicator molecules of aflatoxin toxigenic fungi and application of rapid detection kit

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Application publication date: 20140514